Analysis of ras gene mutations and methylation state in human leukemias.

We have screened a large series of primary human leukemias for activating point mutations at codons 12, 13 and 61 of the N-ras and K-ras proto-oncogenes and at codons 12 and 61 of the H-ras proto-oncogene by using panels of oligonucleotide probes in conjunction with polymerase chain reaction gene amplification. 13 of 64 (20%) acute lymphoblastic leukemia cases had ras gene mutations mostly involving N-ras codon 12/13, G-A (gly-asp) transitions. Consistent with previous studies, a comparable pattern and frequency of ras mutation was found amongst 45 cases of acute myeloid leukemia and myelodysplasia. By contrast, of 30 cases of mature B cell chronic lymphocytic leukemia, only one in terminal prolymphocytoid transformation harboured an activated ras gene. These patterns of mutation did not correlate with ras gene methylation state, a finding not obviously compatible with differential gene accessibility being an important determinant of ras gene mutation patterns in leukemogenesis. Our data suggest that activated ras is more important in tumourigenesis of immature than mature lymphocyte progenitors whilst similar mechanisms associated with aetiology and/or target cell susceptibility probably underlie the similar patterns of ras gene mutations seen in acute leukemias of both myeloid and lymphoid cell lineages.

Prevalence of factor V Leiden and prothrombin variant G20210A in patients age <50 years with no significant stenoses at angiography three to four weeks after myocardial infarction.

OBJECTIVES: We sought to determine the frequencies of factor V Leiden and prothrombin variant G20210A in patients age <50 years with no significant coronary stenoses three to four weeks after myocardial infarction (MI). BACKGROUND: Factor V Leiden and prothrombin variant G20210A occur frequently in patients with venous thromboembolism. However, the contribution of these mutations to the development of MI requires clarification. METHODS: The frequencies of factor V Leiden and prothrombin variant G20210A were determined in 41 patients age <50 years who had "normal" or "near normal" coronary arteries (no stenosis >50%) at angiography three to four weeks after MI (the study group) and compared with those in 114 patients who had at least one angiographic stenosis >50% after MI (the control group). Patients age > or =50 years with, or without, stenoses were also studied. RESULTS: The frequency of factor V Leiden was 14.6% in patients age <50 years in the study group compared with 3.6% in patients in the control group (odds ratio [OR] 4.7 [95% confidence interval (CI) 1.3-17.7], p = 0.02). The frequency of the prothrombin variant G20210A was 7.3% in the study group compared with 1.8% in the control group (OR 4.4 [95% CI 0.7-27.5], p = 0.12). One or both mutations were present in 8 of the 41 patients (19.5%) age <50 years in the study group compared with 6 of the 114 patients (5.5%) in the control group (OR 4.4 [95% CI 1.4-13.5], p = 0.01). In all 271 patients (irrespective of age) with normal arteries, the frequency of factor V Leiden was 11.7% (7/60) compared with 4.3% (9/211) in patients with at least one >50% stenosis (OR 2.9 [95% CI 1.1-8.3], p = 0.04), and the frequency of prothrombin variant G20210A was 6.7% (4/60) compared with 1.4% (3/211) (OR 4.9 [95% CI 1.1-22.8], p = 0.04), respectively. CONCLUSIONS: The frequencies of factor V Leiden and/or prothrombin variant G20210A are increased in patients age <50 years with normal or near normal coronary arteries after MI.

Activation of Harvey ras oncogene by mutation at codon 12 is very rare in hemopoietic malignancies.

Point mutations within codon 12 of the Harvey (H-) ras proto-oncogene have recently been implicated in the progression of hemopoietic malignancy, particularly chronic myeloid leukemia. We have analyzed DNA from 170 cases of acute and chronic leukemia by using a restriction fragment length polymorphism. No evidence for clonal allelic H-ras codon 12 activation was found among these cases, which included 23 cases of chronic myeloid leukemia, 12 of which were in accelerated phase or blastic transformation. These data suggest that H-ras codon 12 mutations occur infrequently in hemopoietic neoplasms generally and may be less important in disease progression than has been previously suggested.

Colorectal neoplasms detected colonoscopically in at-risk members of colorectal cancer families stratified by the demonstration of DNA microsatellite instability.

This study compared colonoscopic findings in families meeting the Amsterdam criteria (A) for hereditary non-polyposis colorectal cancer (HNPCC) but stratified according to whether the familial cancers showed DNA microsatellite instability. DNA was extracted from paired samples of normal and cancer, and microsatellite instability was analysed at up to six loci. Families were termed replication error positive (RER+) when at least 50% of tumours tested per family were positive. Of 26 families studied 17 were RER+ and 9 were RER-. Cancers in the A/RER- families showed no right-sided predilection (P < 0.001). Colonoscopies have been performed on 182 at-risk members of A/RER+ families and 60 members of A/RER- families. More of the at-risk members of A/RER-families were found to have adenomas at colonoscopy (P = 0.095), but these were smaller than those of A/RER+ families (P = 0.19). The adenoma:carcinoma ratio was twice as high in A/RER- families (13:1) as in A/RER+ families (7:1). One of the A/RER- families had hyperplastic polyposis. The others do not appear to have attenuated familial adenomatous polyposis and are similar to the adenoma families or late-onset colorectal cancer families described by others. This study illustrates the importance of molecular technology in separating HNPCC from syndromes with overlapping phenotypes.

Mixed acute leukaemias.

In recent years immunophenotyping and analysis of clonal rearrangement of immunoglobulin and T-cell antigen receptor genes have proved valuable for the diagnosis and classification of leukaemia. These techniques aid in the assignment of cell lineage in cases of acute leukaemia in which the standard FAB criteria of morphology and cytochemistry do not reveal clear lymphoid or myeloid phenotype. These new techniques have also revealed that the leukaemic blasts in a sizable minority of otherwise typical cases of acute leukaemia express 'inappropriate' lineage-associated markers and have been termed mixed acute leukaemias. The spectrum of characteristics encompassed by mixed acute leukaemias ranges from fairly common cases expressing one or two inappropriate markers to the more extreme, rare cases of acute leukaemia termed 'hybrid' in which a truly scrambled picture is seen. A subgroup of these mixed cases have two distinct populations of blasts, e.g. one lymphoid and the other myeloid. These observations raise a number of issues about the cell of origin of these leukaemias and about the mechanisms controlling the developmental regulation of expression of different lineage-associated markers. In addition, accumulating evidence suggests that inappropriate expression of markers may identify sub-groups of both acute myeloid and lymphoblastic leukaemia with an inferior prognosis.

Use of the X-chromosome linked hypoxanthine phosphoribosyl transferase gene as a marker of cell monoclonality in hemopoietic malignancies.

The X-chromosome linked gene encoding hypoxanthine phosphoribosyl transferase (HPRT) has been reported to provide a novel approach to the investigation of cell monoclonality in females based on non-random methylation-sensitive restriction enzyme cleavage of the active vs inactive HPRT alleles and a polymorphic Bam HI restriction site to distinguish the maternal and paternal gene copies. In a survey of 80 females, which included 65 cases of hemopoietic malignancy and 15 normal individuals, we found only nine (11.3%) to be heterozygous (informative) for the Bam HI polymorphism. Monoclonality was demonstrable by HPRT gene analysis in five of six informative cases of leukemia, the exception being a case of acute myeloid leukemia which displayed anomalous methylation of the HPRT gene. Our studies suggest that the applicability of the HPRT gene probe strategy may be limited by (1) the low frequency of informative cases and (2) potential inappropriate methylation of the HPRT gene in a proportion of cases.

Absence of Kirsten-ras oncogene activation in B-cell chronic lymphocytic leukemia.

By using a combination oligonucleotide probe hybridization and restriction enzyme polymorphism analysis, a series of 48 cases of B-cell chronic lymphocytic leukemia were investigated for activating point mutations at codons 12, 13 and 61 of the K-ras proto-oncogene. A small series of acute leukemias (seven with acute lymphoblastic leukemia (ALL), 11 with acute myeloid leukemia (AML)) were examined in parallel. None of the cases of B-CLL contained detectable activating mutations of the K-ras gene at codon 12 (GGT-gly----GCT-ala) was detected at presentation. In both cases of acute leukemia, the mutation was restricted to one allele and could not be detected in remission samples. Those data suggest that activation of members of the ras oncogene family, typified by K-ras, may be less important in disease pathogenesis in leukemias such as B-CLL that arise from a more committed progenitor.

Successful treatment of veno-occlusive disease with recombinant tissue plasminogen activator in a patient requiring peritoneal dialysis.

There have been encouraging reports of the use of recombinant tissue plasminogen activator (tPA) in established veno-occlusive disease (VOD). Haemodialysis has been considered a contraindication to this therapy in view of the potential haemostatic complications. We report a case of a woman who developed moderately severe VOD complicated by anuria following an allogeneic bone marrow transplant for relapsed acute myeloid leukaemia. Following initiation of peritoneal dialysis she received tPA at a dose of 10 mg/day for 5 days. There was rapid improvement in her urine output and liver function with no bleeding complications. This case suggests that the requirement of dialysis may not preclude the use of tPA in established VOD and therefore warrants further study.

Graft-versus-host disease following interferon therapy for relapsed chronic myeloid leukaemia post-allogeneic bone marrow transplantation.

The combination of donor leucocytes, with or without interferon, has produced encouraging responses in patients with haematological relapse following allogeneic BMT for chronic myeloid leukaemia (CML). A 25-year-old male received low-dose interferon-alpha alone for haematological relapse occurring 10 months following an allogeneic BMT for Ph-positive CML. Interferon therapy was complicated by severe GVHD requiring immunosuppressive therapy. The patient was subsequently found to be in complete haematological and cytogenetic remission, raising the possibility of an immune-mediated antileukaemic action.

Prolonged remission of Epstein-Barr virus associated lymphoma secondary to T cell-depleted bone marrow transplantation.

High grade non-Hodgkin's lymphoma developed 42 days after allogeneic T cell-depleted bone marrow transplantation (BMT) for idiopathic aplastic anaemia. DNA hybridization studies confirmed clonality and incorporation of Epstein-Barr virus (EBV) genome. Prolonged remission followed low dose chemotherapy, local radiotherapy and early withdrawal of cyclosporin.

Deletion of the long arm of chromosome 20 in a patient with small cell lymphocytic lymphoma.

Cytogenetic analysis of bone marrow from an 80-year-old woman with small cell lymphocytic lymphoma revealed a del(20)(q11.2) as the sole cytogenetic abnormality. Del(20q) is found almost exclusively in myeloid disorders and this case represents a rare occurrence in a non-myeloid disorder.

Chromosome 22 breakpoints in variant Philadelphia translocations and Philadelphia-negative chronic myeloid leukemia.

The standard t(9;22)(q34;q11) found in Philadelphia (Ph) chromosome positive chronic myeloid leukemia (CML) involves a highly restricted (5.8 kb) chromosome 22 breakpoint cluster region (bcr), which results in the formation of a chimeric gene comprising exons from the 5' end of bcr and protooncogene c-abl coding sequences from chromosome 9. In a survey of 21 patients with hematologic and clinical features of CML we detected rearrangement of the chromosome 22 bcr by gene probe analysis in all cases, including 16 with a standard t(9;22), two with variant Ph translocations [t(10;22)(q26;q11);t(11;22)(p15;q11)], one with a complex Ph translocation [t(9;11;22)(q34;q13;q11)], one with a complex translocation and a masked Ph[t(9;14;22) (q34;q24;q11)], and one Ph-negative case with a t(1;9)(p32;q34). These observations further substantiate the suggestion that, despite karyotypic heterogeneity, a common underlying molecular lesion, the bcr-abl gene chimera, is involved in the disease pathogenesis of CML.

Transformation of essential thrombocythaemia to T cell acute lymphoblastic leukaemia.

Leukaemic transformation of essential thrombocythaemia is a rare event and is usually associated with previous treatment with either alkylating agents or radioactive phosphorous. We describe a patient with essential thrombocythaemia who developed an acute leukaemia of T cell phenotype following hydroxyurea therapy. The T cell phenotype of the blasts suggests the target cell for leukaemic transformation was a pluripotential stem cell.

Factor VIII gene inversions in severe hemophilia A patients.

The mutations causing hemophilia A are very heterogeneous with the exception of a large inversion involving intron 22 in the factor VIII (FVIII) gene which appears to be the underlying defect in approximately 45% of all severely affected patients (FVIII < or = 1%). In these patients it is thought that the factor VIII gene is disrupted within intron 22 due to inappropriate recombination of FVIIIA with one of 2 homologous regions upstream of the factor VIII gene resulting in a large (approximately 500 kb) inversion. The inversion can be detected by Southern blot analysis and greatly enhances the accuracy of genetic counselling services available to families with severe hemophilia A. We report here the presence of this mutation in a study of 27 unrelated families with severe hemophilia. The factor VIII inversion was identified in 12 of 27 (44%) severe hemophilia A patients and has been successfully used for direct carrier analysis and prenatal diagnosis.

Pseudo-lymphocyte monocytes--the memory cells responsible for the development of epitheloid cell granulomata. A new hypothesis.

This hypothesis proposes that, in individuals with an appropriate genetic background, monocyte memory cells are formed when histiocytes and macrophages undergo mitosis following first exposure to a granulomagenic agent and circulate as pseudolymphocytes in the lymphocyte null cell population. It is proposed that epithelioid cell granulomata develop from a clone of cells formed from monocyte memory cells on the second or subsequent exposure to the same granulomagenic agent. Epithelioid cell granuloma formation is therefore not dependent on T-cell function, although the cellular nature of the granuloma appears to depend upon the nature of a concomitant but independent classical immune response. The implications of pseudolymphocyte memory cells on the development of granulomata of both exogenous and endogenous origin, and the relationships between lymphocytes and cells of the monocytic phagocyte series are discussed.

Haemophilia management: the application of DNA analysis for prenatal diagnosis.

The haemophilias are chronic debilitating disorders which cause significant morbidity for the patient and may affect the whole family. An important part of the management of these disorders is the provision of accurate carrier detection and prenatal diagnosis in conjunction with genetic counselling services. We report the results of carrier detection and prenatal diagnosis obtained over a two year period using recombinant DNA techniques. Eighty-seven individuals from 15 families with either haemophilia A or B have been evaluated using informative intragenic factor VIII or IX restriction fragment length polymorphisms. Chorionic villi biopsies for prenatal diagnosis have been performed in four subjects, revealing two female carriers, one normal male, one normal of unknown sex and one haemophiliac male. The use of genotypic diagnosis of haemophilia A and B, in conjunction with conventional assays, is now a routine part of the modern management of haemophilia and many other inherited disorders.

Experimental granulomatous inflammation: the ultrastructure of the granuloma induced by injection of tubercle bacilli into Freund adjuvant-sensitised guinea-pigs.

Injection of killed tubercle bacilli into a sensitised guinea pig produces a characteristic biphasic response with the development of an organised epithelioid cell granuloma in the second phase. Previous sensitisation to tubercle bacilli is a requirement for development of the organised granulomatous response. The main components of the granuloma are epithelioid cells, although multinucleate cells of both Langhans and foreign-body type are present. Epithelioid cells appear to evolve from monocytes, probably in the sequence: (a) monocyte, (b) monocytic transition form, (c) immature epithelioid cell, (d) mature epithelioid cell, although some may possibly develop through a macrophage stage. Differentiation of monocytes into epithelioid cells is a continual process in the experimental tuberculous granuloma with monocytes migrating into the lesion at all stages examined. Epithelioid cells are not obviously phagocytic. Their differ4ntiation has a phase suggestive of biosynthesis during which RER is the predominant cytoplasmic component. This is followed by a storage/secretory phase in which the cytoplasm contains membrane-lined vesicles and prominent Golgi apparatus. The vesicles and, where distended, the RER laminae contain a lightly staining, finely granular material the biological activity of which is unknown.

Experimental granulomatous inflammation: the ultrastructure of the reaction of guinea pigs to bentonite injection.

Bentonite, a silicate, induces a classical non-immunological foreign body reaction when injected intradermally into guinea pigs. The cellular response consists of a mass of macrophages and large macrophage polykaryons, surrounded and infiltrated by an extensive fibrous reaction. The bentonite granuloma shows no signs of intercellular organisation of the reacting cells. Study of its ultrastructure suggests a low turnover lesion with a stable, long-lived cell population. The bentonite granuloma is contrasted with the tuberculous, immunologically mediated epithelioid cell granuloma produced in sensitised guinea pigs.